Respiratory syncytial virus expression vectors

ABSTRACT

In certain embodiments, the disclosure relates to vectors containing bacterial nucleic acid sequences and a paramyxovirus gene. Typically, the expression vector comprises a bacterial artificial chromosome (BAC), and a nucleic acid sequence comprising a respiratory syncytial virus (RSV) gene in operable combination with a regulatory element and optionally a reporter gene.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/474,365 filed Apr. 12, 2011, hereby incorporated by reference in itsentirety.

This invention was made with government support under Grant NoUL1RR025008 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Human respiratory syncytial virus (RSV) causes respiratory tractinfections. It is the major cause of hospital visits during infancy andchildhood. After translation of viral mRNAs, a full-length (+)antigenomic RNA is produced as a template for replication of the (−) RNAgenome. Infectious recombinant RSV (rRSV) particles may be recoveredfrom transfected plasmids. Co-expression of RSV N, P, L, and M2-1proteins as well as the full-length antigenomic RNA is sufficient forRSV replication. See Collins et al., Proc Natl Acad Sci USA., 1995,92(25):11563-11567 and U.S. Pat. No. 6,790,449.

Despite the existence of methods of generating RSV particles from clonedcDNA, stability of RSV cDNA remains a challenge. A region of the RSVsmall hydrophobic protein (SH) gene is unstable as cloned cDNA. Seee.g., Skiadopoulos et al., Virology 345, 492-501 (2006). Investigatorshave suffered failures in cloning RSV cDNA in plasmids, despiteextensive experience with other viruses and cDNA cloning. Labs typicallyuse a RSV antigenomic cDNA cloned in the plasmid pBR322. In order tomaintain the antigenomic cDNA in this plasmid, one typically grows thebacteria at 30° C. and low aeration. Nevertheless, plasmids frequentlyundergo rearrangements and clone loss. Taken together, plasmid stabilityis a factor limiting progress in RSV research and vaccine development.Thus, there is a need to identify improved methods of generating RSV.

One may recover viruses from bacterial artificial chromosome (BAC)vectors. See Roth et al., Vet Res., 2011, 42(1):3 and Alder et al., RevMed. Virol., 2003, 13(2):111-21 and U.S. Pat. No. 7,892,822. BACrecombineering refers to a method of introducing mutations in cDNAscloned in a BAC vectors via homologous recombination in E coli. A BACrecombineering system based on selection and counter-selection of thegalK operon was disclosed by Warming et al, Nucleic Acids Research,2005, 33, e36. References cited herein are not an admission of priorart.

SUMMARY

In certain embodiments, the disclosure relates to vectors comprising abacterial artificial chromosome (BAC), and a nucleic acid sequencecomprising a paramyxovirus genome, antigenome, or gene of aparamyxovirus. Typically, the paramyxovirus is respiratory syncytialvirus (RSV), human metapneumovirus, nipah virus, hendra virus, orpneumonia virus and the BAC contains all genes that are essential forthe generation of an infectious viral particle in a host cell. Thenucleic acid sequence may be a viral genome or antigenome in operablecombination with a regulatory element. Typically, the bacterialartificial chromosome comprises one or more genes selected from thegroup consisting of oriS, repE, parA, and parB genes of Factor F inoperable combination with a selectable marker, e.g., a gene thatprovides resistance to an antibiotic.

The nucleic acid sequence may be the genomic or antigenomic sequence ofthe virus which is optionally mutated, e.g., RSV strain which isoptionally mutated. In certain embodiments, the expression vector is aplasmid comprising MluI, ClaI, BstBl, SacI restriction endonucleasecleavage sites and optionally an AvrII restriction endonuclease cleavagesite outside the region of the wild-type viral sequence or outside thesequences that encode viral genes or outside the viral genome orantigenome. In certain embodiments, the nucleic acid sequence furthercomprises a selectable marker or reporter gene in operable combinationtherewith, e.g., a gene that encodes a fluorescent protein.

In certain embodiments, the disclosure relates to isolated bacteriacomprising one or more vectors disclosed herein, and other embodiments,the disclosure relates to an isolated cell comprising one or morevectors disclosed herein. In certain embodiments, the vector comprisesan RSV antigenome and one or more vectors selected from the groupconsisting of: a vector encoding an N protein of RSV, a vector encodinga P protein of RSV, a vector encoding an L protein of RSV, and a vectorencoding an M2-1 protein of RSV. Typically, the vector comprises aregulatory element, e.g., promoter, and the isolated eukaryotic cellexpresses a nucleic acid or polypeptide that activates the regulatoryelement, e.g., encodes a polypeptide that activates transcriptiondownstream of the promoter. In certain embodiments, the promoter is T7,and the polypeptide that activates transcription downstream of thepromoter is T7 RNA polymerase.

In certain embodiments, the disclosure relates to methods of generatingrespiratory syncytial virus (RSV) particles comprising inserting avector with a BAC gene and a RSV antigenome into an isolated eukaryoticcell and inserting one or more vectors selected from the groupconsisting of: a vector encoding an N protein of RSV, a vector encodinga P protein of RSV, a vector encoding an L protein of RSV, and a vectorencoding an M2-1 protein of RSV into the cell under conditions such thatRSV particle is formed. Inserting a vector into a cell may occur byphysically injecting, electroporating, or mixing the cell and the vectorunder conditions such that the vector infects the cell.

In certain embodiments, the disclosure relates to a non-naturallyoccurring isolated nucleic acid comprising or consisting essential ofSEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 or a sequence withsubstantial identity.

In certain embodiments, the disclosure relates to a non-naturallyoccurring isolated nucleic acid comprising or consisting essential ofSEQ ID NO: 4 and SEQ ID NO: 5 or a sequence with substantial identity.

In certain embodiments, the disclosure relates to a recombinant vectorcomprising a bacterial artificial chromosome, a nucleic acid sequencecomprising SEQ ID NO: 4 or a sequence with substantial identity; and anucleic acid sequence comprising SEQ ID NO: 5 or a sequence withsubstantial identity.

In certain embodiment, the disclosure relates to processes of producinga recombinant vector comprising a bacterial artificial chromosome andSacI, ClaI and AvrII restriction endonuclease cleavage sites comprisingmixing a nucleic acid comprising a bacterial artificial chromosome and anucleic acid comprising SacI, ClaI and AvrII restriction endonucleasecleavage sites under conditions such that a continuous nucleic acidcomprising a bacterial artificial chromosome and a SacI, ClaI and AvrIIrestriction endonuclease cleavage sites is formed.

In certain embodiments, the disclosure relates to a recombinant vectorcomprising SEQ ID NO: 6 or a sequence with substantial identity.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 shows a gel after insertion of galK operon into BAC-RSV byrecombineering. MluI digest. Lane 1, ladder marker. Mini-prep BAC DNAs(lanes 2 to 7). Lane 8, parental BAC-RSV “C2” clone. Lane 9,galK-containing plasmid. galK operon has a Mlu I restriction site thatserves as a marker for introduction of galK by homologous recombination.

FIG. 2 shows a gel after deletion of galK operon from BAC-RSV byrecombineering. MluI digest of galK-containing plasmid (lane 2), BACmini-prep DNAs (lanes 3-7), and parental BAC-RSV clone C2 (lane 8).

FIGS. 3A-E schematically illustrate steps for creating a BAC-RSV. Threeplasmids with RSV segments are generated (see experimental); A) pKBS3 iscut at BstBl and Mlul sites to linearize, and is ligated to anoligonucleotide adapter providing pKBS5; B) pSynRSV#2 with SacI and ClaIis cut and ligated to pKBS5 providing pKBS5-2; C) pSynRSV#3 with AvrIIand Mlul is cut and ligated to pKBS5_(—)2 providing pKBS5_(—)2_(—)3; D)pSynRSV#1 with BstB1 and SacI is cut and ligated to pKBS5_(—)2_(—)3providing pKBS5_(—)1_(—)2_(—)3. E). Recombineering is used to deletenucleotides between two ClaI sites generating pSynRSV-line 19F.

DETAILED DESCRIPTION

It has been discovered that cultivating RSV in E. coli bacteria may beaccomplished by utilizing a plasmid containing a bacterial artificialchromosome. A plasmid comprising a bacterial artificial chromosome isdisclosed that contains the complete antigenomic sequence of respiratorysyncytial virus (RSV) strain A2 except the F gene, which is theantigenomic sequence of RSV strain line 19. Along with helper plasmids,it can be used in the reverse genetics system for the recovery ofinfectious virus. The antigenome sequence on the plasmid can be mutatedprior to virus recovery to generate viruses with desired mutations.

The plasmid is an improvement on current RSV antigenomic plasmids forseveral reasons. Each RSV gene is flanked by restriction endonucleasecleavage sites to allow for easy manipulation of any gene. As a basisfor viral mutagenesis, this plasmid may be used to design attenuatedviruses for use in vaccines. An extra gene encoding the modifiedkatushka, mKate2, protein has been included in the antigenome prior tothe first RSV gene. Katushka is a red fluorescent protein which would beexpressed in concert with the other RSV genes and would serve as visualevidence of virus replication. Changes have also been made to theribozyme sequences that flank the RSV antigenome and play a role in theproduction of infectious virus through reverse genetics.

The disclosed vectors allow for efficient mutagenesis throughrecombineering. This mutagenesis method requires little to no ligationcloning, but relies on the recombination machinery present in bacteriaharboring certain genes from a bacteriophage. Because RSV genes areoften unstable in bacteria predominantly used for cloning, such asEschericha coli (E. coli), it is believed that the single digit copynature of the bacterial artificial chromosome avoids the trouble withinstability.

Respiratory Syncytial Virus (RSV)

Typically, the RSV particle contains a viral genome within a helicalnucleocapsid which is surrounded by matrix proteins and an envelopecontaining glycoproteins. The genome of human wild-type RSV encodes theproteins, NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L. G, F, and SHare glycoproteins. The F gene has been incorporated into a number ofviral vaccines. RSV polymerase activity consists of the large protein(L) and phosphoprotein (P). The viral M2-1 protein is used duringtranscription and is likely to be a component of the transcriptasecomplex. The viral N protein is used to encapsidate the nascent RNAduring replication.

The genome is transcribed and replicated in the cytoplasm of a hostcell. Host-cell transcription typically results in synthesis of tenmethylated and polyadenylated mRNAs. The antigenome is positive-senseRNA complement of the genome produced during replication, which in turnacts as a template for genome synthesis. The viral genes are flanked byconserved gene-start (GS) and gene-end (GE) sequences. At the 3′ and 5′ends of the genome are leader and trailer nucleotides. The wild typeleader sequence contains a promoter at the 3′ end. When the viralpolymerase reaches a GE signal, the polymerase polyadenylates andreleases the mRNA and reinitiates RNA synthesis at the next GS signal.The L-P complex is believed to be responsible for recognition of thepromoter, RNA synthesis, capping and methylation of the 5′ termini ofthe mRNAs and polyadenylation of their 3′ ends. It is believed that thepolymerase sometimes dissociates from the gene at the junctions. Becausethe polymerase initiates transcription at the 3′ end of the genome, thisresults in a gradient of expression, with the genes at the 3′ end of thegenome being transcribed more frequently than those at the 5′ end.

To replicate the genome, the polymerase does not respond to thecis-acting GE and GS signals and generates positive-sense RNA complementof the genome, the antigenome. At the 3′ end of the antigenome is thecomplement of the trailer, which contains a promoter. The polymeraseuses this promoter to generate genome-sense RNA. Unlike mRNA, which isreleased as naked RNA, the antigenome and genome RNAs are encapsidatedwith virus nucleoprotein (N) as they are synthesized.

In certain embodiments, the disclosure relates to vectors and nucleicacids that contain RSV gene(s) such as the wild-type genome orantigenome. An example of an RSV antigenome is provided in U.S. Pat. No.6,790,449, as SEQ ID NO:1 therein, hereby incorporated by reference.Reference to RSV gene(s) and the genome is contemplated to includecertain mutations, deletions, or variant combinations, such ascold-passaged (cp) non-temperature sensitive (ts) derivatives of RSV,cpRSV, such as rA2 cp248/404/1030ASH. rA2 cp248/404ASH contains 4independent attenuating genetic elements: cp which is based on 5missense mutations in the N and L proteins and the F glycoprotein thattogether confer the non-ts attenuation phenotype of cpRSV; ts248, amissense mutation in the L protein; ts404, a nucleotide substitution inthe gene-start transcription signal of the M2 gene; and ASH, completedeletion of the SH gene. rA2 cp248/404/1030ASH contains 5 independentattenuating genetic elements: those present in rA2 cp248/404ASH andts1030, another missense mutation in the L protein. See Karron et al., JInfect Dis., 2005, 191(7): 1093-1104, hereby incorporated by reference.Within certain embodiments, it is contemplated that the RSV anitgenomemay contain deletion or mutations in nonessential genes (e.g., the SH,NS1, NS2, and M2-2 genes) or combinations thereof.

It is contemplated that the nucleic acid may contain a viral genomeother than RSV which includes an F gene of RSV such as live-attenuatedvaccines, e.g., sendai virus (a murine parainfluenza virus) basedvaccine or a live-attenuated chimeric bovine/human with humanparainfluenza virus vaccine, genetically engineered to express human RSVF protein.

Bacterial Artificial Chromosomes (BACs)

In certain embodiments, the disclosure relates to vectors and nucleicacids that contain bacterial artificial chromosomes. A bacterial cloningsystem for mapping and analysis of complex genomes has been disclosed inShizuya et al., Proc. Natl. Acad. Sci., 1992, 89:8794-8797. The BACsystem (for bacterial artificial chromosome) is based on Escherichiacoli and its single-copy plasmid F factor which were described as usefulfor cloning large fragments of human DNA. The F factor encodes for genesthat regulate its own replication including oriS, repE, parA, and parB.The oriS and repE genes mediate the unidirectional replication of the Ffactor while parA and parB typically maintain copy number at a level ofone or two per E. coli genome. It is contemplated that the genes and thechromosome may contain mutations, deletions, or variants with desiredfunctional attributes. The BAC vector (pBAC) typically contains thesegenes as well as a resistance marker and a cloning segment containingpromotors for incorporating nucleic acid segments of interest byligating into restriction enzyme sites. Exemplary BAC systems includethose described in Shizuya & Kouros-Hehr, Keio J Med, 2001, 50(1):26-30, hereby incorporated by reference.

One may reconstitute infectious RSV virus from the RSV BAC plasmidsdisclosed herein. BAC vectors can be transfected to bacteria such as E.coli by electroporation. The RSV-BACs disclosed herein may be stablymaintained in bacteria, re-isolated from the bacteria, and inserted intoa eukaryotic cell along with one or more vectors that express the N, P,L, and M2-1 proteins. These cells produce infective RSV particles.Production of infectious RSV results from co-transfection of plasmidsencoding N, P, L, and M2-1 proteins and the antigenome under control ofthe T7 promoter into BHK-21 cells that express T7 RNA polymerase (BSRcells). See Buchholz et al., J. Virol., 2000, 74(3):1187-1199, herebyincorporated by reference.

Vaccines

A number of attenuated RSV strains as candidate vaccines for intranasaladministration have been developed using multiple rounds of chemicalmutagenesis to introduce multiple mutations into a virus. Evaluation inrodents, chimpanzees, adults and infants indicate that certain of thesecandidate vaccine strains are immunogenic, and may be attenuated.Nucleotide sequence analysis of some of these attenuated virusesindicates that each level of increased attenuation is typicallyassociated with two or more new nucleotide and amino acid substitutions.

The disclosure provides the ability to distinguish between silentincidental mutations versus those responsible for phenotype differencesby introducing the mutations, separately and in various combinations,into the genome or antigenome of infectious wild-type RSV. This processidentifies mutations responsible for phenotypes such as attenuation,temperature sensitivity, cold-adaptation, small plaque size, host rangerestriction, etc. Mutations from this menu can then be introduced invarious combinations to calibrate a vaccine virus to an appropriatelevel of attenuation, etc., as desired. Moreover, the present disclosureprovides the ability to combine mutations from different strains ofvirus into one strain.

The present disclosure also provides for methods of attenuation. Forexample, individual internal genes of human RSV can be replaced withtheir bovine, murine or other RSV counterpart. This may include part orall of one or more of the NS1, NS2, N, P, M, SH, M2-1, M2-2 and L genes,or parts of the G and F genes. Reciprocally, means are provided togenerate a live attenuated bovine RSV by inserting human attenuatinggenes into a bovine RSV genome or antigenome background. Human RSVbearing bovine RSV glycoproteins provides a host range restrictionfavorable for human vaccine preparations. Bovine RSV sequences which canbe used in the present disclosure are described in, e.g., Pastey et al.,J. Gen. Viol. 76:193-197 (1993); Pastey et al., Virus Res. 29:195-202(1993); Zamora et al., J. Gen. Virol. 73:737-741 (1992); Mallipeddi etal., J. Gen. Virol. 74:2001-2004 (1993); Mallipeddi et al., J. Gen.Virol. 73:2441-2444 (1992); and Zamora et al., Virus Res. 24:115-121(1992), each of which is incorporated herein by reference.

The disclosure also provides the ability to analyze other types ofattenuating mutation and to incorporate them into infectious RSV forvaccine or other uses. For example, a tissue culture-adaptednonpathogenic strain of pneumonia virus of mice (the murine counterpartof RSV) lacks a cytoplasmic tail of the G protein (Randhawa et al.,Virology 207: 240-245 (1995)). By analogy, the cytoplasmic andtransmembrane domains of each of the RSV glycoproteins, F, G and SH, canbe deleted or modified to achieve attenuation.

Other mutations for use in infectious RSV of the present disclosureinclude mutations in cis-acting signals identified during mutationalanalysis of RSV minigenomes. For example, insertional and deletionalanalysis of the leader and trailer and flanking sequences identifiedviral promoters and transcription signals and provided a series ofmutations associated with varying degrees of reduction of RNAreplication or transcription. Saturation mutagenesis (whereby eachposition in turn is modified to each of the nucleotide alternatives) ofthese cis-acting signals also has identified many mutations whichreduced (or in one case increased) RNA replication or transcription. Anyof these mutations can be inserted into the complete antigenome orgenome as described herein. Other mutations involve replacement of the3′ end of genome with its counterpart from antigenome, which isassociated with changes in RNA replication and transcription. Inaddition, the intergenic regions (Collins et al., Proc. Natl. Acad. Sci.USA 83:4594-4598 (1986), incorporated herein by reference) can beshortened or lengthened or changed in sequence content, and thenaturally-occurring gene overlap (Collins et al., Proc. Natl. Acad. Sci.USA 84:5134-5138 (1987), incorporated herein by reference) can beremoved or changed to a different intergenic region by the methodsdescribed herein.

In another embodiment, RSV useful in a vaccine formulation can beconveniently modified to accommodate antigenic drift in circulatingvirus. Typically the modification will be in the G and/or F proteins.The entire G or F gene, or the segment(s) encoding particularimmunogenic regions thereof, is incorporated into the RSV genome orantigenome cDNA by replacement of the corresponding region in theinfectious clone or by adding one or more copies of the gene such thatseveral antigenic forms are represented. Progeny virus produced from themodified RSV cDNA are then used in vaccination protocols against theemerging strains. Further, inclusion of the G protein gene of RSVsubgroup B would broaden the response to cover a wider spectrum of therelatively diverse subgroup A and B strains present in the humanpopulation.

An infectious RSV clone of the disclosure can also be engineered toenhance its immunogenicity and induce a level of protection greater thanthat provided by natural infection, or vice versa, to identify andablate epitopes associated with undesirable immunopathologic reactions.Enhanced immunogenicity of the vaccines produced by the presentdisclosure addresses one of the greatest obstacles to controlling RSV,namely the incomplete nature of immunity induced by natural infection.An additional gene may be inserted into or proximate to the RSV genomeor antigenome which is under the control of an independent set oftranscription signals. Genes of interest include those encodingcytokines (e.g., IL-2 through IL-15, especially IL-3, IL-6 and IL-7,etc.), gamma-interferon, and proteins rich in T helper cell epitopes.The additional protein can be expressed either as a separate protein oras a chimera engineered from a second copy of one of the RSV proteins,such as SH. This provides the ability to modify and improve the immuneresponse against RSV both quantitatively and qualitatively.

For vaccine use, virus produced according to the present disclosure canbe used directly in vaccine formulations, or lyophilized, as desired,using lyophilization protocols well known to the artisan. Lyophilizedvirus will typically be maintained at about 4 degrees C. When ready foruse the lyophilized virus is reconstituted in a stabilizing solution,e.g., saline or comprising SPG, Mg, and HEPES, with or without adjuvant,as further described below.

Thus RSV vaccines of the disclosure contain as an active ingredient animmunogenetically effective amount of RSV produced as described herein.The modified virus may be introduced into a host with a physiologicallyacceptable carrier and/or adjuvant. Useful carriers are well known inthe art, and include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, hyaluronic acid and the like. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration, as mentioned above. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, and the like. Acceptable adjuvants include incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum, which arematerials well known in the art.

Upon immunization with a RSV composition as described herein, viaaerosol, droplet, oral, topical or other route, the immune system of thehost responds to the vaccine by producing antibodies specific for RSVvirus proteins, e.g., F and G glycoproteins. As a result of thevaccination the host becomes at least partially or completely immune toRSV infection, or resistant to developing moderate or severe RSVinfection, particularly of the lower respiratory tract.

The host to which the vaccine are administered can be any mammal whichis susceptible to infection by RSV or a closely related virus and whichhost is capable of generating a protective immune response to theantigens of the vaccinizing strain. Thus, suitable hosts include humans,non-human primates, bovine, equine, swine, ovine, caprine, lagamorph,rodents, etc. Accordingly, the invention provides methods for creatingvaccines for a variety of human and veterinary uses.

The vaccine compositions containing the RSV of the disclosure areadministered to a host susceptible to or otherwise at risk of RSVinfection to enhance the host's own immune response capabilities. Suchan amount is defined to be an “immunogenically effective dose.” In thisuse, the precise amounts again depend on the host's state of health andweight, the mode of administration, the nature of the formulation. Thevaccine formulations should provide a quantity of modified RSV of theinvention sufficient to effectively protect the host patient againstserious or life-threatening RSV infection.

The RSV produced in accordance with the present invention can becombined with viruses of the other subgroup or strains to achieveprotection against multiple RSV subgroups or strains, or protectiveepitopes of these strains can be engineered into one virus as describedherein. Typically the different viruses will be in admixture andadministered simultaneously, but may also be administered separately.For example, as the F glycoproteins of the two RSV subgroups differ byonly about 11% in amino acid sequence, this similarity is the basis fora cross-protective immune response as observed in animals immunized withRSV or F antigen and challenged with a heterologous strain. Thus,immunization with one strain may protect against different strains ofthe same or different subgroup.

In some instances it may be desirable to combine the RSV vaccines of thedisclosure with vaccines which induce protective responses to otheragents, particularly other childhood viruses. For example, the RSVvaccine of the present disclosure can be administered simultaneouslywith parainfluenza virus vaccine, such as described in Clements et al.,J. Clin. Microbiol. 29:1175-1182 (1991), incorporated herein byreference. In another aspect of the disclosure the RSV can be employedas a vector for protective antigens of other respiratory tractpathogens, such as parainfluenza, by incorporating the sequencesencoding those protective antigens into the RSV genome or antigenomewhich is used to produce infectious RSV as described herein.

Single or multiple administrations of the vaccine compositions of thedisclosure can be carried out. In neonates and infants, multipleadministration may be required to elicit sufficient levels of immunity.Administration should begin within the first month of life, and atintervals throughout childhood, such as at two months, six months, oneyear and two years, as necessary to maintain sufficient levels ofprotection against native (wild-type) RSV infection. Similarly, adultswho are particularly susceptible to repeated or serious RSV infection,such as, for example, health care workers, day care workers, familymembers of young children, the elderly, individuals with compromisedcardiopulmonary function, may require multiple immunizations toestablish and/or maintain protective immune responses. Levels of inducedimmunity can be monitored by measuring amounts of neutralizing secretoryand serum antibodies, and dosages adjusted or vaccinations repeated asnecessary to maintain desired levels of protection. Further, differentvaccine viruses may be advantageous for different recipient groups. Forexample, an engineered RSV strain expressing an additional protein richin T cell epitopes may be particularly advantageous for adults ratherthan for infants.

In yet another aspect of the disclosure, the RSV is employed as a vectorfor transient gene therapy of the respiratory tract. According to thisembodiment, the recombinant RSV genome or antigenome incorporates asequence which is capable of encoding a gene product of interest. Thegene product of interest is under control of the same or a differentpromoter from that which controls RSV expression. The infectious RSVproduced by coexpressing the recombinant RSV genome or antigenome withthe N, P, L and M2-1 proteins and containing a sequence encoding thegene product of interest is administered to a patient. Administration istypically by aerosol, nebulizer, or other topical application to therespiratory tract of the patient being treated. Recombinant RSV isadministered in an amount sufficient to result in the expression oftherapeutic or prophylactic levels of the desired gene product. Examplesof representative gene products which are administered in this methodinclude those which encode, for example, those particularly suitable fortransient expression, e.g., interleukin-2, interleukin-4,gamma-interferon, GM-CSF, G-CSF, erythropoietin, and other cytokines,glucocerebrosidase, phenylalanine hydroxylase, cystic fibrosistransmembrane conductance regulator (CFTR), hypoxanthine-guaninephosphoribosyl transferase, cytotoxins, tumor suppressor genes,antisense RNAs, and vaccine antigens.

TERMS

The terms “protein” and “polypeptide” refer to compounds comprisingamino acids joined via peptide bonds and are used interchangeably.

The term “portion” when used in reference to a protein (as in “a portionof a given protein”) refers to fragments of that protein. The fragmentsmay range in size from four amino acid residues to the entire aminosequence minus one amino acid.

The term “chimera” when used in reference to a polypeptide refers to theexpression product of two or more coding sequences obtained fromdifferent genes, that have been cloned together and that, aftertranslation, act as a single polypeptide sequence. Chimeric polypeptidesare also referred to as “hybrid” polypeptides. The coding sequencesinclude those obtained from the same or from different species oforganisms.

The term “homolog” or “homologous” when used in reference to apolypeptide refers to a high degree of sequence identity between twopolypeptides, or to a high degree of similarity between thethree-dimensional structure or to a high degree of similarity betweenthe active site and the mechanism of action. In a preferred embodiment,a homolog has a greater than 60% sequence identity, and more preferablygreater than 75% sequence identity, and still more preferably greaterthan 90% sequence identity, with a reference sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

The terms “variant” and “mutant” when used in reference to a polypeptiderefer to an amino acid sequence that differs by one or more amino acidsfrom another, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions (inother words, additions), or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Preferred variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or a polypeptide or its precursor (e.g., proinsulin). A functionalpolypeptide can be encoded by a full length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the polypeptide are retained. The term “portion”when used in reference to a gene refers to fragments of that gene. Thefragments may range in size from a few nucleotides to the entire genesequence minus one nucleotide. Thus, “a nucleotide comprising at least aportion of a gene” may comprise fragments of the gene or the entiregene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences. The term“gene” encompasses both cDNA and genomic forms of a gene. A genomic formor clone of a gene contains the coding region interrupted withnon-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are segments of a gene which aretranscribed into nuclear RNA (mRNA); introns may contain regulatoryelements such as enhancers. Introns are removed or “spliced out” fromthe nuclear or primary transcript; introns therefore are absent in themessenger RNA (mRNA) transcript. The mRNA functions during translationto specify the sequence or order of amino acids in a nascentpolypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, posttranscriptionalcleavage and polyadenylation.

The term “heterologous gene” refers to a gene encoding a factor that isnot in its natural environment (i.e., has been altered by the hand ofman). For example, a heterologous gene includes a gene from one speciesintroduced into another species. A heterologous gene also includes agene native to an organism that has been altered in some way (e.g.,mutated, added in multiple copies, linked to a non-native promoter orenhancer sequence, etc.). Heterologous genes are distinguished fromendogenous plant genes in that the heterologous gene sequences aretypically joined to nucleotide sequences comprising regulatory elementssuch as promoters that are not found naturally associated with the genefor the protein encoded by the heterologous gene or with plant genesequences in the chromosome, or are associated with portions of thechromosome not found in nature (e.g., genes expressed in loci where thegene is not normally expressed).

The term “polynucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The polynucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. The term “oligonucleotide” generally refers to a short lengthof single-stranded polynucleotide chain usually less than 30 nucleotideslong, although it may also be used interchangeably with the term“polynucleotide.”

The term “nucleic acid” refers to a polymer of nucleotides, or apolynucleotide, as described above. The term is used to designate asingle molecule, or a collection of molecules. Nucleic acids may besingle stranded or double stranded, and may include coding regions andregions of various control elements, as described below.

The term “a polynucleotide having a nucleotide sequence encoding a gene”or “a polynucleotide having a nucleotide sequence encoding a gene” or “anucleic acid sequence encoding” a specified polypeptide refers to anucleic acid sequence comprising the coding region of a gene or in otherwords the nucleic acid sequence which encodes a gene product. The codingregion may be present in either a cDNA, genomic DNA or RNA form. Whenpresent in a DNA form, the oligonucleotide, polynucleotide, or nucleicacid may be single-stranded (i.e., the sense strand) or double-stranded.Suitable control elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. or acombination of both endogenous and exogenous control elements.

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, for the sequence “A-G-T,” is complementary to the sequence“T-C-A.” Complementarity may be “partial,” in which only some of thenucleic acids' bases are matched according to the base pairing rules.Or, there may be “complete” or “total” complementarity between thenucleic acids. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon binding between nucleic acids.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology or completehomology (i.e., identity). “Sequence identity” refers to a measure ofrelatedness between two or more nucleic acids or proteins, and is givenas a percentage with reference to the total comparison length. Theidentity calculation takes into account those nucleotide or amino acidresidues that are identical and in the same relative positions in theirrespective larger sequences. Calculations of identity may be performedby algorithms contained within computer programs such as “GAP” (GeneticsComputer Group, Madison, Wis.) and “ALIGN” (DNAStar, Madison, Wis.). Apartially complementary sequence is one that at least partially inhibits(or competes with) a completely complementary sequence from hybridizingto a target nucleic acid is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a sequence which is completelyhomologous to a target under conditions of low stringency. This is notto say that conditions of low stringency are such that non-specificbinding is permitted; low stringency conditions require that the bindingof two sequences to one another be a specific (i.e., selective)interaction. The absence of non-specific binding may be tested by theuse of a second target which lacks even a partial degree ofcomplementarity (e.g., less than about 30% identity); in the absence ofnon-specific binding the probe will not hybridize to the secondnon-complementary target.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)) by the homology alignmentalgorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol.48:443 (1970)), by the search for similarity method of Pearson andLipman (Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.) 85:2444(1988)), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25 50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low to highstringency as described above.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low to high stringency as described above.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “regulatory element” refers to a genetic element which controlssome aspect of the expression of nucleic acid sequences. For example, apromoter is a regulatory element which facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements are splicing signals, polyadenylation signals, terminationsignals, etc.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237, 1987). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect, mammalian and plant cells.Promoter and enhancer elements have also been isolated from viruses andanalogous control elements, such as promoters, are also found inprokaryotes. The selection of a particular promoter and enhancer dependson the cell type used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review, see Voss,et al., Trends Biochem. Sci., 11:287, 1986; and Maniatis, et al., supra1987).

The terms “promoter element,” “promoter,” or “promoter sequence” as usedherein, refer to a DNA sequence that is located at the 5′ end (i.e.precedes) the protein coding region of a DNA polymer. The location ofmost promoters known in nature precedes the transcribed region. Thepromoter functions as a switch, activating the expression of a gene. Ifthe gene is activated, it is said to be transcribed, or participating intranscription. Transcription involves the synthesis of mRNA from thegene. The promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA.

Promoters may be tissue specific or cell specific. The term “tissuespecific” as it applies to a promoter refers to a promoter that iscapable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue (e.g., seeds) in the relativeabsence of expression of the same nucleotide sequence of interest in adifferent type of tissue (e.g., leaves). Tissue specificity of apromoter may be evaluated by, for example, operably linking a reportergene to the promoter sequence to generate a reporter construct,introducing the reporter construct into the genome of a plant such thatthe reporter construct is integrated into every tissue of the resultingtransgenic plant, and detecting the expression of the reporter gene(e.g., detecting mRNA, protein, or the activity of a protein encoded bythe reporter gene) in different tissues of the transgenic plant. Thedetection of a greater level of expression of the reporter gene in oneor more tissues relative to the level of expression of the reporter genein other tissues shows that the promoter is specific for the tissues inwhich greater levels of expression are detected. The term “cell typespecific” as applied to a promoter refers to a promoter which is capableof directing selective expression of a nucleotide sequence of interestin a specific type of cell in the relative absence of expression of thesame nucleotide sequence of interest in a different type of cell withinthe same tissue. The term “cell type specific” when applied to apromoter also means a promoter capable of promoting selective expressionof a nucleotide sequence of interest in a region within a single tissue.Cell type specificity of a promoter may be assessed using methods wellknown in the art, e.g., immunohistochemical staining Briefly, tissuesections are embedded in paraffin, and paraffin sections are reactedwith a primary antibody which is specific for the polypeptide productencoded by the nucleotide sequence of interest whose expression iscontrolled by the promoter. A labeled (e.g., peroxidase conjugated)secondary antibody which is specific for the primary antibody is allowedto bind to the sectioned tissue and specific binding detected (e.g.,with avidin/biotin) by microscopy.

Promoters may be constitutive or regulatable. The term “constitutive”when made in reference to a promoter means that the promoter is capableof directing transcription of an operably linked nucleic acid sequencein the absence of a stimulus (e.g., heat shock, chemicals, light, etc.).Typically, constitutive promoters are capable of directing expression ofa transgene in substantially any cell and any tissue.

In contrast, a “regulatable” or “inducible” promoter is one which iscapable of directing a level of transcription of an operably linkednucleic acid sequence in the presence of a stimulus (e.g., heat shock,chemicals, light, etc.) which is different from the level oftranscription of the operably linked nucleic acid sequence in theabsence of the stimulus.

The enhancer and/or promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer or promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer or promoter is one that is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of the gene isdirected by the linked enhancer or promoter. For example, an endogenouspromoter in operable combination with a first gene can be isolated,removed, and placed in operable combination with a second gene, therebymaking it a “heterologous promoter” in operable combination with thesecond gene. A variety of such combinations are contemplated (e.g., thefirst and second genes can be from the same species, or from differentspecies).

Efficient expression of recombinant DNA sequences in eukaryotic cellstypically requires expression of signals directing the efficienttermination and polyadenylation of the resulting transcript.Transcription termination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly(A) tail areunstable and are rapidly degraded. The poly(A) signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly(A) signal is one that is found naturally at the 3′ end of thecoding region of a given gene in the genome. A heterologous poly(A)signal is one which has been isolated from one gene and positioned 3′ toanother gene. A commonly used heterologous poly(A) signal is the SV40poly(A) signal. The SV40 poly(A) signal is contained on a 237 byBamHI/BclI restriction fragment and directs both termination andpolyadenylation.

The term “vector” refers to nucleic acid molecules that transfer DNAsegment(s) from one cell to another. The term “vehicle” is sometimesused interchangeably with “vector.”

The terms “expression vector” or “expression cassette” refer to arecombinant nucleic acid containing a desired coding sequence andappropriate nucleic acid sequences used for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences used for expression in prokaryotes typically include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

The term “host cell” refers to any cell capable of replicating and/ortranscribing and/or translating a heterologous gene. Thus, a “host cell”refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells suchas E. coli, yeast cells, mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo. For example, host cells may be located in a transgenic animal.

The term “selectable marker” refers to a gene which encodes an enzymehaving an activity that confers resistance to an antibiotic or drug uponthe cell in which the selectable marker is expressed, or which confersexpression of a trait which can be detected (e.g., luminescence orfluorescence). Selectable markers may be “positive” or “negative.”Examples of positive selectable markers include the neomycinphosphotrasferase (NPTII) gene which confers resistance to G418 and tokanamycin, and the bacterial hygromycin phosphotransferase gene (hyg),which confers resistance to the antibiotic hygromycin. Negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene is commonly used as a negative selectablemarker. Expression of the HSV-tk gene in cells grown in the presence ofgancyclovir or acyclovir is cytotoxic; thus, growth of cells inselective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme.

The term “reporter gene” refers to a gene encoding a protein that may beassayed. Examples of reporter genes include, but are not limited to,modified katushka, mkate and mkate2 (See, e.g., Merzlyak et al., Nat.Methods, 2007, 4, 555-557 and Shcherbo et al., Biochem. J., 2008, 418,567-574), luciferase (See, e.g., deWet et al., Mol. Cell. Biol. 7:725(1987) and U.S. Pat Nos., 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from ClonTech Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, beta-galactosidase,alkaline phosphatase, and horse radish peroxidase.

The term “wild-type” when made in reference to a gene refers to a genewhich has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product which has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as used herein as applied to an object refers tothe fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. A wild-type gene is that which is most frequentlyobserved in a population and is thus arbitrarily designated the “normal”or “wild-type” form of the gene. In contrast, the term “modified” or“mutant” when made in reference to a gene or to a gene product refers,respectively, to a gene or to a gene product which displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

The term “antisense” or “antigenome” refers to a nucleotide sequencewhose sequence of nucleotide residues is in reverse 5′ to 3′ orientationin relation to the sequence of nucleotide residues in a sense strand. A“sense strand” of a DNA duplex refers to a strand in a DNA duplex whichis transcribed by a cell in its natural state into a “sense mRNA.” Thusan “antisense” sequence is a sequence having the same sequence as thenon-coding strand in a DNA duplex.

EXPERIMENTAL Example 1 Expression of RSV in Plasmid Designed for LowCopy Number

Infectious recombinant RSV (rRSV) can be recovered from transfectedplasmids. Co-expression of RSV N, P, L, and M2 1 proteins as well as thefull-length antigenomic RNA is sufficient for RSV replication.Infectious RSV may be produced from the co-transfection of plasmidsencoding N, P, L, and M2-1 proteins and the antigenome under control ofthe T7 promoter into BHK-21 cells that stably express T7 RNA polymerase(BSR cells). Currently research labs typically use a RSV antigenomiccDNA cloned in the plasmid pBR322 (mid-range copy number, 15-20 copiesper E coli). In order to maintain the antigenomic cDNA in this plasmid,the bacteria is grown at 30° C. and low aeration. Nevertheless, plasmidrearrangements and clone loss is frequently experienced.

A fraction of RSV cDNA containing the attachment glycoprotein (G) andfusion (F) genes of the virus was found to be unclonable in pUC-basedplasmids (500-700 plasmid copies in E coli). This fragment was cloned ina low copy number (approximately 5 copies per E. coli) plasmid calledpLG338-30.5. The plasmid pLG338-30 was developed to increase thestability of cloned lentivirus glycoproteins. Cunningham et al., Gene,1993, 124, 93-98. It is hypothesized that cDNA instability in E coliresults from the presence of cryptic E coli transcription promoterswithin viral glycoprotein sequences. Thus, instability of cDNA in“promoterless” plasmids in bacteria can arise because aberrant proteinsare expressed from cryptic promoters, leading to toxicity exacerbated byplasmid copy number.

An antigenomic plasmid was generated containing the RSV strain A2 genomewith the strain line 19 F gene in place of the A2 F gene. It had beenderived from the antigenome plasmid first disclosed in Collins et al.,Proc Natl Acad Sci USA., 1995, 92(25):11563-11567 and U.S. Pat. No.6,790,449 hereby incorporated by reference. The antigenome was digestedout of the plasmid vector and ligated into the pKBS3 BAC.

GalK recombineering reagents were obtained from the NCI and successfullyestablished a BAC-RSV reverse genetics protocol (FIGS. 1 and 2). Seehttp://web.ncifcrf.gov/research/brb/recombineeringInformation.aspx,hereby incorporated by reference. Mutation of RSV cDNA via BACrecombineering has enhanced the ability to manipulate RSV for generationof mutants. An added benefit of the system is enhanced stability of thefull-length antigenomic cDNA in the BAC vector.

The BAC-based RSV antigenome vector was propagated at 32° C. and 250 RPMwithout observing any vector rearrangements or clone loss in E coli.Thus, BAC-RSV not only enables manipulations via recombineering but alsofacilitates RSV reverse genetics in general owing to elimination of cDNAinstability.

Example 2 RSV Antigenome in BAC Vector (pSynkRSV_line 19 F Construction)

The RSV-BAC pSynkRSV_line 19 F contains the modified katushka gene(mKate2, fluorescent protein), and restriction sites for more convenientcloning. To build pSynkRSV, three nucleic acid pieces were synthesizedby Gene Art, a German company that synthesizes DNA. These three piecesthen have to be put together in the bacterial artificial chromosome(BAC). The three pieces are designated pSynkRSV-BstBI_SacI (#1) (SEQ IDNO: 1), pSynkRSV-SacI_ClaI (#2) (SEQ ID NO: 2), and pSynkRSV-ClaI_MluI(#3) (SEQ ID NO: 3). One uses the plasmid pKBS3 as the backbone forconstructing pSynkRSV. See FIGS. 3A-E. pSynkRSV contains the bacterialartificial chromosome sequences needed to regulate copy number andpartitioning in the bacteria.

To insert the three synthesized segments, one puts oligonucleotideadapters into pKBS3 between two existing restriction enzyme cut sites,BstBI and MluI.

(SEQ ID NO: 4) CGAATTGGGAGCTCTTTTATCGATGTTGCCTAGGTTTTA (SEQ ID NO: 5)TTAACCCTCGAGAAAATAGCTACAACGGATCCAAAATGCGC

The overhangs were designed such that the adapter would ligate intopKBS3 at the BstBI and MluI sites. Underlined sequences indicaterestriction sites: SacI, ClaI, and AvrII from right to leftrespectively. This produces a multi-cloning site containing therestriction sites BstBI, SacI, ClaI, AvrII, and MluI, in that order, anda plasmid termed pKBSS. See FIG. 3A. One cuts and ligates the SacI ClaIsegment (#2) from Gene Art into pKBSS. See FIG. 3B. Next one cuts andligates the #3 segment using the enzymes AvrII and MluI (cannot use ClaIagain due to an inactive ClaI restriction site in pSynkRSV-ClaI_MluI).See FIG. 3C. At this point, the plasmid pKBSS contains the Gene Artsequences from SacI to ClaI, some intervening nucleotides (less than10), and the Gene Art sequences from AvrII to MluI. One cuts and ligatesthe #1 segment using BstBI and SacI. See FIG. 3D. This RSV BAC containsabout 10 unwanted nucleotides between two ClaI sites (that from segment#2 and segment #3). Recombineering is used to delete those nucleotides,thus generating pSynkRSV_line 19 F (SEQ ID NO: 6). See FIG. 3E. Thethree segments should be ligated in this order to avoid potentialinterference from multiple restriction sites.

1. A vector comprising a) a bacterial artificial chromosome, and b) anucleic acid sequence comprising a gene of a paramyxovirus.
 2. Thevector of claim 1, wherein the paramyxovirus is respiratory syncytialvirus (RSV), human metapneumovirus, nipah virus, hendra virus, orpneumonia virus.
 3. The vector of claim 1, wherein the nucleic acidsequence comprising a gene of a paramyxovirus is the genomic orantigenomic sequence of RSV.
 4. The vector of claim 1, wherein thenucleic acid sequence comprising a gene of a paramyxovirus is an RSVstrain that is optionally mutated.
 5. The vector of claim 1, wherein thebacterial artificial chromosome comprises a gene selected from the groupconsisting of oriS, repE, parA, and parB genes of Factor F.
 6. Thevector of claim 1, wherein the bacterial artificial chromosome comprisesa selectable marker.
 7. The vector of claim 1, wherein the vector is aplasmid comprising MluI, ClaI, BstBl, SacI restriction endonucleasecleavage sites and optionally a AvrII, restriction endonuclease cleavagesite.
 8. The vector of claim 1, wherein the nucleic acid is arespiratory syncytial virus (RSV) antigenome in operable combinationwith a reporter gene.
 9. An isolated bacteria comprising the vector ofclaim
 1. 10. An isolated cell comprising the vector of claim
 1. 11. Theisolated cell of claim 10, further comprising a vector selected from thegroup consisting of a vector encoding an N protein of RSV, a vectorencoding a P protein of RSV, a vector encoding an L protein of RSV, anda vector encoding an M2-1 protein of RSV.
 12. The isolated cell of claim11, wherein the vector comprises a promoter and the isolated cellencodes a polypeptide that activates transcription downstream of thepromoter.
 13. The isolated cell of claim 12, wherein the promoter is T7.14. The isolated cell of claim 12, wherein the polypeptide thatactivates transcription downstream of the promoter is T7 RNA polymerase.15. A method of generating respiratory syncytial virus (RSV) particlecomprising a) inserting a vector of claim 1 into an isolated eukaryoticcell, and b) inserting a vector selected from the group consisting of avector encoding an N protein of RSV, a vector encoding a P protein ofRSV, a vector encoding an L protein of RSV, and a vector encoding anM2-1 protein of RSV into the cell under conditions such that RSVparticle is formed.
 16. An non-naturally occurring isolated nucleic acidconsisting essential of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:
 3. 17.An non-naturally occurring isolated nucleic acid consisting essential ofSEQ ID NO: 4 and SEQ ID NO:
 5. 18. A vector comprising a) a bacterialartificial chromosome; b) a nucleic acid sequence comprising SEQ ID NO:4; and c) a nucleic acid sequence comprising SEQ ID NO:
 5. 19. Arecombinant vector comprising SEQ ID NO: 6.